The primary objectives of this trial were to describe the compartmental pharmacokinetics of meloxicam in calves after IV administration at 0.5 mg/kg and to determine the analgesic effect of meloxicam after scoop dehorning and thermocautery without local anesthesia. Surgery-induced pain and central sensitization consist of two phases: an immediate incisional phase and a prolonged inflammatory phase that arises primarily due to tissue damage . Demonstrating the adequacy of preemptive analgesia has two basic requirements . The first is to demonstrate the direct pharmacological effect of the analgesic. This was accomplished in the present study by comparing differences in acute biomarkers of pain and distress including substance P, cortisol response and heart rate between treated and control subjects. The second requirement is to demonstrate the extension of the antinociceptive effect into the postoperative period when pain due to inflammation becomes the dominant process . In practical terms, two approaches have been used to demonstrate the efficacy of preemptive analgesic regimens. The first is to demonstrate a reduction in pain intensity beyond the presence of the drug in the biophase in studies involving treated and untreated control subjects. The second approach is to demonstrate that a treatment applied before surgery is more effective than the treatment applied at the end of surgery . In the present study, we used pharmacokinetic analysis to determine the presence of meloxicam in the biophase and cortisol, SP and heart rate analysis to determine the direct pharmacological effect of meloxicam in treated and untreated calves. Continuous, telemetric assessment of posture and activity over 96 hours after dehorning and weight gain over 10 days was used to determine if meloxicam effects extended into the post-operative period.
In the present study, plasma meloxicam concentrations were still quantifiable in the last sample, at 52 hours post-injection, and showed a bi-exponential decay following administration. The initial steep decline in plasma concentrations was likely due to the rapid distribution of the drug from the central to the peripheral compartment. This was followed by a slower decline in plasma concentrations associated with drug metabolism and excretion. Although exceptions due to species and drug compound exist, NSAIDs in general tend to be highly protein-bound in the plasma which limits distribution into the tissue, leading to low volume of the central compartment and low volume of distribution  as was seen in the current study. The extended half-life of meloxicam in cattle is likely due to a low total body clearance representing mostly hepatic clearance since high levels of protein binding tend to limit glomerular filtration of drug compounds.
The clinical implication of the slow elimination of meloxicam from the body is that infrequent drug administration (once every few days) may be sufficient to mitigate pain effects due to post-surgical inflammation in calves. It was recently reported that generic meloxicam tablets have 100% bioavailability following oral administration in ruminant calves suggesting that this may provide a practical and cost effective alternative to IV administration in those animals . The pharmacokinetic profile of meloxicam described in the current report along with the associated effects on behavior and performance suggest that administration immediately prior to dehorning may have effects for several days post-dehorning. Given that the plasma half-life of meloxicam is longer than previously reported for ketoprofen (0.42 h) , salicylate (0.5 h)  and flunixin (4- 8 h) , these results suggest that meloxicam may have an extended duration of activity compared with other NSAIDs currently available in the U.S. However, further research is needed to determine if the effect of these analgesics is directly related to plasma drug concentrations, and if so, to determine the effective range. Although the literature is deficient in studies with cattle, the effective plasma concentration (EC50) of meloxicam is estimated to be 0.73 μg/mL in the horse  and 0.36 μg/mL in the dog . If cattle respond to meloxicam as horses and dogs, the effective plasma drug concentration would be maintained for several days following IV administration of meloxicam at 0.5mg/kg.
Cortisol, substance P and heart rate analysis was used as an indicator of the direct pharmacological effect of meloxicam on pain and distress associated with dehorning in treated and untreated calves. It has been reported that plasma cortisol concentrations reach a peak within 30 minutes after dehorning after which levels decrease to a plateau concentration that persists for 5 – 6 hours [19, 20]. The results presented here are consistent with previous reports that demonstrate a significant increase in plasma cortisol concentration after dehorning [4, 5, 21]. However, in contrast with the findings of Heinrich and others , the present study failed to demonstrate that calves receiving meloxicam prior to dehorning had lower cortisol concentrations compared with untreated controls. This may be due to differences in the blood collection schedule that was designed to minimize the effect of animal handling on behavioral assessment. Furthermore, the present study enrolled older calves (<3mo vs. >4 mo) and necessarily employed a different method of dehorning (cautery disbudding vs. amputation). Since the horn bud attaches to the skull of calves at approximately 2 months of age, then gradually develops a diverticulum communicating with the frontal sinus, removal of the horn tissue is generally considered more invasive and stressful in older animals resulting in higher cortisol concentrations .
The most likely explanation for the differences in cortisol response is that previous reports frequently combined NSAID administration with local anesthesia. Local anesthetics mitigate acute incisional pain by blocking voltage-gated sodium channels in the nerves preventing the generation and propagation of nerve impulses or action potentials . In the bovine, this effect lasts approximately 2 – 3 h after dehorning with the provision of a lidocaine block of the cornual nerve [10, 20]. Lidocaine local anesthesia combined with systemic ketoprofen administration prior to dehorning significantly attenuates the plasma cortisol response compared with the effect of the agents administered separately [19, 20, 23]. The reason why we chose not to administer local anesthesia in the present study was because surveys suggest that less that 20% of U.S. dairy producers currently use local anesthetics prior to dehorning and our goal was to look at the effect of an NSAID without local anesthesia [1, 8, 9]. The absence of an effect of meloxicam on acute cortisol response suggests that provision of systemic analgesia alone without local anesthesia is inadequate in providing adequate preemptive analgesia using the definition provided by Kissin, 2000 .
Substance P is an 11-amino acid prototypic neuropeptide that regulates the excitability of dorsal horn nociceptive neurons and is expressed in areas of the neuroaxis involved in the integration of pain, stress, and anxiety . It has been reported that plasma SP concentrations are significantly higher in beef calves after castration compared with uncastrated controls . In the present study, mean plasma substance P concentration was estimated to be reduced by 50% (95% Confidence interval: 26 to 96%) in calves that received meloxicam prior to dehorning compared with placebo-treated controls. Furthermore, we observed that increases in plasma substance P concentrations from baseline corresponded with lower log plasma meloxicam concentrations. These findings support the hypothesis that meloxicam treatment mitigates SP release and therefore potentially reduced pain perception in calves after dehorning without local anesthesia. To our knowledge this is the first study that has demonstrated a relationship between substance P concentrations and analgesic drug concentrations after dehorning. This suggests that SP measurement may have potential to be used as a biomarker for determining analgesic drug efficacy in calves subjected to painful procedures. Furthermore, the absence of an effect of meloxicam treatment on serum cortisol concentrations suggests that simultaneous determination of plasma SP and cortisol concentrations during painful procedures may assist in differentiating between acute stress associated with handling and distress associated with nociception as previously described .
Normal HR in unstressed cattle range from 70 to 90 bpm  but mean HR has been shown to increase by 30 to 40 bpm over baseline levels by stressful events such as branding, electric shock and handling prior to dehorning and castration [21, 27, 28]. Schwartzkopf-Genswein and others reported that HR in dehorned calves was significantly higher that control animals for 120 minutes after the procedure . Similarly, Grondahl-Nielsen et al. (1999) observed that HR was elevated for 3.5 h in dehorned calves receiving no anesthetic or analgesic compared with calves that were only sham dehorned . Likewise, Stewart et al, 2009 observed that HR was raised above baseline for 3 h after dehorning without local anesthesia . Heinrich et al (2009) observed a greater increase in HR, measured by thoracic auscultation at 7 time points over 24 h, in placebo-treated calves compared with calves that received 0.5 mg/kg meloxicam IM combined with corneal nerve block at 10 minutes prior to dehorning . In the present study, baseline HR was higher than previously reported and were maintained above 100 bpm for 6 hours after dehorning in both treatment groups. This difference could be attributed to the use of older calves (16 – 20 wks vs. < 6 wks) than what had been used in previous experiments. In our study we observed a significant decrease in mean HR in meloxicam-treated calves at 8 and 10 hours after dehorning. These results support the conclusions of Heinrich and others (2009)  that meloxicam reduces the stress response after dehorning as reflected by in changes in heart rate even in the absence of local anesthetic administration at the time of dehorning.
Comparison between calf behavior before and after dehorning and assessment of weight gain over 10 days post-dehorning was used to determine if meloxicam effects extended into the post-operative period. It was recently reported that calves that received oral meloxicam at 1 mg/kg spent more time lying down over 4 days compared with placebo-treated control calves . The amount of time cattle exhibit specific behaviors is commonly used to indicate comfort and/or clinical illness [31–34]. An increase or decrease in lying behavior, however, does not expressly indicate pain or comfort and must be interpreted within the context of what is normal for a particular animal. Cattle in pain due to lameness have been observed to lie more  whereas cattle in pain due to dehorning and castration have been observed to lie less than nonpainful controls [6, 30, 36, 37]. Furthermore, lying behavior among individual cattle within a herd has been observed to vary more than lying behavior between herds . Therefore, within-animal comparisons, such as implicitly occurs with an analysis such as in the current study comparing an individual’s control period (pre-procedure) and treatment period (post-procedure) behavior, are likely to be more sensitive than between-animal comparisons in detecting changes due to a particular treatment. Since meloxicam-treated calves were intermingled equally with placebo-treated calves, the difference noted between groups in the pre-procedure period is likely due to individual variation in the normal amount of time spent lying.
It has been previously reported that calves that received meloxicam at 0.5 mg/kg IM combined with corneal nerve blocks were less active than controls during the first 5 h following dehorning compared with placebo-treated controls . In the present study, calves receiving meloxicam without local anesthesia demonstrated no significant difference in lying activity before and after dehorning. In contrast, control calves spent less time lying after dehorning, and although the percentage difference was numerically small (3.4%), this difference was significant due to the associated small standard error. This is similar to the results reported by Theurer and others (2012) that observed differences in lying behavior for 5 days after dehorning in calves that received oral meloxicam at 1 mg/kg . When considering a 3.4% difference over a 24 hour period, control calves stood an average of 49 minutes more per day after dehorning than before. In meloxicam calves, the absence of an effect of dehorning on lying behavior may have been due to the analgesic activity of the drug. These results suggest that meloxicam mitigates behavioral effects of dehorning even in the absence of local anesthetic administration.
Studies reporting a difference in weight gain between analgesic-treated and unmedicated calves after dehorning are deficient in the published literature. Previously, Faulkner and Weary (2000) demonstrated that calves receiving ketoprofen prior to dehorning tended to gain more weight during the 24 h after dehorning than untreated calves . However, during the subsequent 24 hour period, weight gains were similar between the two groups. Administration of the NSAID, sodium salicylate in drinking water for 2 d after dehorning and castration was found to increase ADG over 13 d . Over the 10 day duration of the present study, calves receiving meloxicam gained significantly more weight than those in the control group, with a mean difference of 0.65 ± 0.28 kg/day (p=0.0418). This finding supports the hypothesis that extended exposure to a non-steroidal anti-inflammatory drug (NSAID) may maintain growth and performance after castration.
Although studies reporting a performance benefit after NSAID administration 2- 3 weeks following processing and castration are deficient in the published literature, meloxicam administration has been associated with improved average daily gain in calves suffering from clinical bovine respiratory disease . Therefore, in order to definitively attribute the observed difference in ADG to the effect of meloxicam on pain and inflammation associated only with dehorning, future studies should also include additional meloxicam treated and untreated control calves that are processed but not dehorned.
The biological explanation for the improved ADG observed in this study was not investigated. Meloxicam treated and untreated control calves were co-mingled in pens for the duration of the study precluding assessment of individual animal feed intake or feed efficiency. Furthermore, even though the accelerometer analysis revealed significant differences in the pre and post-dehorning behavior of control calves, this technology is currently not sufficiently sensitive to characterize subtle differences in feeding and walking behavior that could contribute to performance differences. However, Theurer and others  recently reported that calves that received oral meloxicam prior to dehorning spent more time at the grain bunk and less time at the hay feeder compared to the control group which could explain the difference in weight gain observed in the present study. Another possible contributory factor to the performance difference is that increased activity of nociceptors increases sympathetic tone and adrenal secretory activity which may inhibit gastric centers causing decreased rumen motility . Mellor and others (2002) reported a significant increase in plasma adrenaline and noradrenaline concentration for 5 and 50 minutes respectively after scoop dehorning in 10 week old calves . Adrenergic influences on reticuloruminal motility comprise depression of the gastric centers resulting in inhibition of intrinsic and extrinsic rumen motility . The duration of these effects and how these relate to plasma catecholamine concentrations has not been reported. Future studies examining individual or pen level feed intake and feed efficiency and the association with rumen motility after dehorning may help explain the difference in ADG observed in this report.